Sudden cardiac death (SCD) is a major public health problem responsible for up to 63% of cardiac deaths among persons older than 35 in the United States.1 While acute ischemic heart disease is the major cause of SCD in this population, one of the most common causes of sudden death in adolescents and young adults less than 40 years old is hypertrophic cardiomyopathy (HCM). Other less frequent causes include anomalous coronary arteries, Marfan's syndrome, arrhythmogenic right ventricular dysplasia, myocarditis, aortic valvular stenosis and coronary atherosclerotic disease. HCM is an inherited disease of cardiac muscles where there is asymmetric or symmetric left ventricular hypertrophy with a non-dilated, hyperdynamic chamber in the absence of secondary causes. It affects men and women equally and is characterized by highly variable clinical courses, heterogeneous pathology and complex genetic expression. Depending on underlying mutations, HCM can be compatible with normal longevity at one end of disease spectrum, but at the other end, it is the most common cause of SCD in young athletes. The causes of SCD are assumed to be complex ventricular tachycardia or fibrillation originating on an electrically unstable myocardial substrate, but bradycardias and severe obstruction are also suggested. Prevention of SCD in HCM continues to be a major challenge for clinicians. The use of implantable cardioverter defibrillator (ICD) in tertiary centers has been reported to be useful, but the patients may still suffer from end-stage heart failure and atrial fibrillation with attendant risk of thromboembolic strokes.3,4 A pathological model of HCM will be critical to understand the development of HCM and causes of SCD, and that will be important for prevention and treatment. Currently, the known disease genes account for about 50-70% of all cases of familial hypertrophic cardiomyopathy.5 Most of the primary cardiomyopathies in humans, hypertrophic or dilated, are due to mutations in contractile proteins, cytoskeletal proteins, or gap junction proteins, and are in most cases the direct consequences of structural defects.
The cardiac extracellular matrix plays multiple important functional roles as the substrate for cell adhesions and scaffolding, as the signals for cell survival, as a reservoir for growth factors, and as the major determinant of tissue mechanics. Collagen is a major component of extracellular matrix in the mammalian hearts and exhibits a dynamic expression pattern and isoform diversity during development and in pathological conditions.5 In the adult myocardium, type I collagen comprises 80% of total collagen and together with type III collagen forms a network of fibrillar collagen that supports individual cardiomyocytes and smooth muscle cells, and organizes muscle bundles, conduction system, and coronary vasculature into cohesive contractile units. In contrast, type III collagen is the major component in neonatal rat hearts, comprising about one-third of the total collagen content, and thus the visco-elasticity is different from that of adult myocardium. In rodent models of right ventricular hypertrophy, the ratio of types I and III collagen is clearly altered.9, 10 Types II and VIII collagen are transiently expressed during cardiac development and have been implicated in cardiac morphogenesis. Although type II collagen was classically thought to be cartilage specific, a much wider tissue distribution has been shown during mouse embryogenesis. The mouse α1(II) collagen gene (Col2a-1) is transiently expressed in many non-cartilaginous tissues such as the notochord, neural retina, tail tendon, heart, surface ectoderm, calvarial mesenchyme, fetal brain, the sensory epithelium of the inner ear and the apical ectodermal ridge of the forming limb buds (AER). Alternative splicing of exon 2 produces type II procollagen mRNAs that either include (IIA mRNA) or exclude (IIB mRNA) this exon. Type IIA mRNA is abundant in cells of the epimyocardium in both the atria and ventricles of 9.5-day embryos but rapidly diminishes by 10.5 days. After 12.5 days, IIB mRNA levels increase rapidly and finally exceed IIA mRNAs. By 16.5 days, IIB mRNAs are the major Col2a-1 transcripts, predominantly expressed in maturing chondrocytes.13 Postmortem analyses from one hospital revealed that defects in cardiac septation were present in 2 infants with hypochondrogenesis, a disorder of type II collagen.
How type IIA collagen contributes to cardiac morphogenesis is not yet clearly defined, but recent studies suggest that it may modulate growth factors. Of note, exon2 of the type II collagen gene (Col2a-1), which is included in IIA mRNA, encodes a cysteine-rich globular domain in the amino-propeptide of pro-α1(II) collagen chains. This cysteine-rich domain has been shown to bind bone morphogenic proteins (eg. BMP2) and Transforming Growth Factor (TGF)-β1 in in vitro assays.
TGF-β1 and BMP-2 belong to the transforming growth factor-β superfamily that regulates growth and differentiation. Like other members of the TGF-β superfamily (TGF-β/Activin/Nodal and BMP/GDF/MIS subfamilies), their diverse biological effects are mediated by the formation of heteromeric complexes of type I and type II serine/threonine kinase receptors, phosphorylation of the type I receptor by type II receptor and subsequent activation of Smad proteins. Receptor-activated Smads (R-Smads, Smads 1-3, 5, 8) are phosphorylated by type I receptor kinases, form heteromeric complexes with the common mediator Smads (co-Smads, Smad4) and translocate into the nucleus where they interact with specific transcription factors and co-regulators to modulate gene expression. R-Smads2 and 3 respond to the TGF-β subfamily, while R-Smads I, 5, and 8 primarily to the BMP subfamily. Two inhibitory Smads (I-Smad), Smad6 and Smad7, negatively regulate TGF-β/BMP signaling by competing with Smad4 for the binding of activated R-Smads, inhibiting phosphorylation of R-Smads, or targeting the receptors for degradation.16, 17 Both TGF-β1 and BMP-2 play crucial roles in various aspects of heart development, especially in cardiomyocyte differentiation, valvulogenesis, and outflow tract and septal development. 18-21 TGF-β1 exerts diverse biological activities in postnatal hearts. It controls the expression of major histocompatibility complex, induces production of the extracellular matrix proteins by cardiac fibroblasts, regulates vascular smooth muscle cell phenotype, induces cardiac hypertrophy, and enhances β-adrenergic signaling. There is limited information about the role of BMP-2 in postnatal mammalian hearts. In rat neonatal cardiomyocyte culture, BMP-2 exerts anti-apoptotic effect by activation of the Smad1 pathway and enhances contractility by activation of phosphatidylinositol 3 kinase pathway.22, 23 Clinically, primary pulmonary hypertension, a disorder of increased proliferation of endothelial and smooth muscle cells in pulmonary arteries, has been linked to mutations in type II receptors for BMP (BMPR-II).24 25 
Recent studies in the murine model of Marfan syndrome also highlight the active role of extracellular matrix, in this instance, fibrillin, in modulating TGF-β signaling.26, 27 
In human dilated cardiomyopathy, increased perimysial fibrosis is associated with increase in types I, III, and VI collagen content, and type V collagen is increased in intracellular matrix of myocardium. Type II collagen is not expressed in normal hearts or dilated cardiomyopathy.28 Whether or not type II collagen is ectopically expressed in hypertrophic cardiomyopathy in humans remains to be investigated. A variety of mouse models of cardiomyopathies and altered contractility focus on targeted mutations of aforementioned proteins, cell cycle regulatory proteins, regulator proteins involved in calcium metabolisms, and subunits of the adrenergic receptor signal transduction cascade. The concept of extracellular matrix modulating tissue patterning and cardiomyocyte differentiation is not novel although matrix defects have not generally been considered as possible source of cardiomyopathies. Even in the mouse with targeted deletion of type III collagen, there was no cardiac abnormality and sudden death was due to rupture of the aorta.29 The adult 191-IIA mutant mice described in this application are therefore a very important model for cardiomyopathy-induced death which may occur suddenly.
Taussig-Bing Anomaly (TBA) is a rare but complex congenital heart disease and comprises of a double outlet right ventricle (DORV) with subpulmonary ventricular septal defect (VSD). Aortic and pulmonary trunks are transposed. Next to Tetralogy of Fallot, TBA is the most common variant of DORV. Infants with TBA often present with congestive heart failure and pulmonary hypertension. Its management is complicated by associated vascular defects such as coronary anomalies, subaortic stenosis, aortic arch coarctation or interruption. Currently recommended treatment is complete correction in a single operation, typically involving arterial switch operation, VSD repair, corrective repair of aortic obstruction, and translocation of anomalous coronary arteries. Therefore, detailed mapping of these vascular defects is crucial to the success of corrective surgery. TOGA is the most frequently diagnosed complex cyanotic heart defects in newborns (ref) and involves ventriculoarterial discordance. Genetics of human TBA or TOGA has focused on candidate genes involved in left-right asymmetry or heterotaxy syndrome. Mutations in three human genes have been associated with TOGA: Zinc-finger transcription factor ZIC3 {Digilio, M. C. et al. Complete transposition of the great arteries: patterns of congenital heart disease in familial precurrence. Circulation 104, 2809-14 (2001).} {Belmont, J. W., Mohapatra, B., Towbin, J. A. & Ware, S. M. Molecular genetics of heterotaxy syndromes. Curr Opin Cardiol 19, 216-20 (2004).}, CFC1 (human CRYPTIC gene) {Goldmuntz, E. et al. CFC1 mutations in patients with transposition of the great arteries and double-outlet right ventricle. Am J Hum Genet 70, 776-80 (2002).}, and PROSIT240 {Muncke, N. et al. Missense mutations and gene interruption in PROSIT240, a novel TRAP240-like gene, in patients with congenital heart defect (transposition of the great arteries). Circulation 108, 2843-50 (2003).}. These clinical findings have been validated in mouse models of genetic ablation of Zic3 {Purandare, S. M. et al. A complex syndrome of left-right axis, central nervous system and axial skeleton defects in Zic3 mutant mice. Development 129, 2293-302 (2002).}, cryptic (EGF-CFC) {Gaio, U. et al. A role of the cryptic gene in the correct establishment of the left-right axis. Curr Biol 9, 1339-42 (1999).}, and TRAP220 {Ito, M., Yuan, C. X., Okano, H. J., Darnell, R. B. & Roeder, R. G. Involvement of the TRAP220 component of the TRAP/SMCC coactivator complex in embryonic development and thyroid hormone action. Mol Cell 5, 683-93 (2000).}.
Here we describe that mice carrying the IIA mutation on a C57/BL6 genetic background is a novel mouse model of TBA and TOGA. Mutations in the human COL2A1 gene are dominant, resulting in chondrodysplasias with a spectrum of severity from prenatal lethality to mild deformity. Dominant mutations in exon2 have been found in the Stickler Syndrome and Wagner's Syndromes which are also associated with eye defects {Richards, A. J. et al. COL2A1 exon 2 mutations: relevance to the Stickler and Wagner syndromes. Br J Ophthalmol 84, 364-71 (2000).} {Van Der Hout, A. H. et al. Occurrence of deletion of a COL2A1 allele as the mutation in Stickler syndrome shows that a collagen type II dosage effect underlies this syndrome. Hum Mutat 20, 236 (2002).} {Donoso, L. A. et al. Identification of a stop codon mutation in exon 2 of the collagen 2A1 gene in a large stickler syndrome family. Am J Ophthalmol 134, 720-7 (2002).} {Donoso, L. A. et al. Clinical variability of Stickler syndrome: role of exon 2 of the collagen COL2A1 gene. Surv Ophthalmol 48, 191-203 (2003).} {Gupta, S. K., Leonard, B. C., Damji, K. F. & Bulman, D. E. A frame shift mutation in a tissue-specific alternatively spliced exon of collagen 2A1 in Wagner's vitreoretinal degeneration. Am J Ophthalmol 133, 203-10 (2002).}. However, no cyanotic congenital heart defects has been reported or systematically correlated with mutations in the Col2a1 gene. Only one postmortem study from one hospital has reported defects in cardiac septation in 2 infants with hypochondrogenesis, a disorder of type II collagen {Potocki, L., Abuelo, D. N. & Oyer, C. E. Cardiac malformation in two infants with hypochondrogenesis. Am J Med Genet 59, 295-9 (1995).}. Given the profound morbidity and mortality of untreated cyanotic congenital heart disease, IIA procollagen may serve as a novel marker for pre-natal screening of complex congenital heart diseases. As a key modulator of BMP-dependent Nkx2.5 expression in cardiac morphogenesis, IIA procollagen may serve as a novel delivery system of growth factors for myocardial regenerative therapy. Understanding the cellular mechanisms of these cardiovascular defects in IIA null mutants will be paramount in formulating a novel strategy to treat or prevent complex cyanotic heart disease.